Influence of the P5 residue on alpha1-proteinase inhibitor mechanism.
The reactive center loop of native alpha1-proteinase inhibitor has been reported to be in a helical conformation and in a beta-strand conformation by two different studies. In the beta-strand loop structure the P5 glutamic acid plays a unique role by stabilizing the loop in the predicted optimal conformation for the interaction with target proteinases and insertion into beta-sheet A. We hypothesize here that disrupting the interactions that stabilize the beta-strand conformation of the loop would result in changes in the inhibitory properties of the serpin. In addition, our earlier studies on reactive center loop mutants of alpha1-proteinase inhibitor suggested that the P5 residue was important in stabilizing the alpha1-proteinase inhibitor-proteinase complexes. To address these issues we made mutants of alpha1-proteinase inhibitor with glycine, glutamine, or lysine at the P5 position and measured the rates and stoichiometries of inhibition with trypsin and human neutrophil elastase and the stabilities of the resulting complexes. In most cases the rate of inhibition was reduced by about half and the stoichiometry increased between 2- and 4-fold. The only exception was for trypsin with the lysine variant where the P5 was now the favored site of cleavage. These data show that the P5 Glu is important in maintaining the reactive center loop in a conformation optimal for interaction with the proteinase and for a fast rate of loop insertion. The complexes formed with trypsin and the variant serpins were less stable than that formed with wild-type serpin and resulted in up to 33% regeneration of trypsin activity over a period of 6 days, compared with 17% with wild type. Thus, the P5 residue of alpha1-proteinase inhibitor is important in all steps of the inhibitory mechanism in a manner consistent with the structural role played by this residue in the beta-strand loop structure of native alpha1-proteinase inhibitor.
Chaillan-Huntington
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CE
Department of Oral Medicine and Diagnostic Sciences and Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Patston
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PA
D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000515:alpha 1-Antitrypsin; D019272:Leukocyte Elastase